Cette recherche a été soutenue par Naval Medical Research Center, unité de recherche de travail 6000.RAD1.J.A0310. Les opinions exprimées dans cet article sont celles des auteurs et ne reflètent pas nécessairement la politique ou la position officielle du ministère de la Marine, Département de la Défense, ou le gouvernement des États-Unis. Wei-Mei Ching est un employé du gouvernement des États-Unis. Ce travail a été préparé dans le cadre de ses fonctions officielles. Titre 17 USC §105 prévoit que «la protection des droits d&#39;auteur sous ce titre ne sont pas disponibles pour tous les travaux du gouvernement des États-Unis.» Titre 17 USC §101 définit un travail du gouvernement des États-Unis comme un travail préparé par un membre du service militaire ou employé du gouvernement américain dans le cadre de fonctions officielles de cette personne.

1. Préparer ADN plasmidique Dilutions comme standard pour LAMP Réaction <ol><li> Utiliser un spectrophotomètre pour mesurer la densité optique (DO) à 260 nm pour déterminer le plasmide (contenant IS1111a du gène cible de C. burnetii RSA 493) la concentration d&#39;ADN. Une DO260 de 1 correspond à 50 pg / pl d&#39;ADN. </li><li> Convertir la quantité d&#39;ADN plasmidique dans le nombre de copies. Étant donné que la taille du plasmide est de 6330 pb, le poids moléculaire de ce plasmide est de 4,1 x 10 6 g (6,330 bp x 649 g / pb). La concentration d&#39;ADN du plasmide est de 34,2 ng / pl (tel que déterminé à l&#39;étape 1.1). 1 ul de cet échantillon d&#39;ADN contient 34,2 ng d&#39;ADN de plasmide, ce qui équivaut à 5 x 10 9 copies (34,2 x 10 -9 g / 4,1 x 10 6 x 10 6,02 gx 23) de l&#39; ADN. </li><li> Ajouter 10 ul d&#39;ADN plasmidique (5 x 10 9 copies / pl) à 90 pl d&#39;eau pour une dilution de 10 fois pour obtenir un échantillon d&#39;ADN de 5 x 10 8 copies / ul. </li><li> Répétez l&#39; étape 1.3 pour préparer des échantillons d&#39; ADN de 5 x 10 7, 5 x 10 6 à 5 x 10 5 5 x 10 4 à 5 x 10 3 à 5 x 10 2 à 5 x 10 1 et 5 x 10 0 copies / ul. </li></ol> 2. Préparer Modèle d&#39;ADN à partir d&#39;échantillons pour LAMP Reaction <ol><li> Réaliser une extraction d&#39;ADN à partir d&#39;échantillons de plasma humain en utilisant un kit d&#39;ADN de mini-utilitaire selon le protocole du fabricant. Utilisez un total de 200 échantillons ul pour l&#39;extraction et éluer l&#39;ADN extrait dans un volume de 20 pi. </li></ol> 3. Préparer 2x tampon de réaction LAMP <ol><li> Mélanger 200 pi de 10x Pol Buffer, 320 pi de 5 M trimethylglycine, 12 pi de 1 M MgSO 4, 280 pi de mélange de dNTP (10 mM chacun) et 188 pi d&#39;eau pour faire 1 ml de tampon 2x LAMP. Mélanger par impulsions tourbillonnement pendant 10 secondes. </li></ol> 4. Effectuez standard LAMP Réaction <ol><li> Mélanger 12,5 pl de 2x LATampon MP (tel que préparé à l&#39; étape 3; 40 mM de Tris-HCl (pH 8,8), KCl 20 mM , du sulfate de magnésium 16 4, 20 mM (NH 4) 2 SO 4, 0,2% de Triton X-100, 1,6 M triméthylglycine, 1,4 mM de mélange de dNTP), 1,2 pi de mélange d&#39; amorces, 1 pi d&#39;ADN polymerase Bst (8 U / ul) et 5,3 ul d&#39;eau pour rendre le mélange réactionnel (20 ul) dans un tube PCR de 0,2 ml. </li><li> Ajouter 5 pi d&#39;ADN (de l&#39;étape 1 ou 2) au mélange de réaction de 20 ul et bien mélanger. </li><li> Fermer le tube et incuber à 60 ° C pendant 60 min dans un bain d&#39;eau ou d&#39;un bloc de chauffage. Ceci est la température de réaction optimale déterminée au préalable. </li><li> Ajouter 5 ul de 10 x tampon de charge de gel pour terminer la réaction. </li><li> Charge 5 pi de produits de réaction sur un gel d&#39;agarose 2% coloré avec intercalation colorant d&#39;acide nucléique. </li><li> Exécutez gel à 100 V pendant 35 min dans un tampon 1x TBE. </li><li> Visualisez les résultats par la lumière UV. </li></ol> 5. Effectuez LAMP Réaction avec ReconstituéRéactifs <ol><li> Mélanger 125 pi de tampon 10x Pol, 200 pi de 5 M triméthylglycine, 7,5 ul de 1 M MgSÛ4, 667,5 pi d&#39;eau pour faire 1 ml de tampon de reconstitution. Mélanger par impulsions tourbillonnement pendant 10 secondes. </li><li> Retirer les tubes qui contiennent les réactifs lyophilisés du sachet en feuille d&#39;aluminium scellée. Ne pas utiliser les tubes si le sac de papier d&#39;aluminium ne soit pas scellé ou si elle est endommagée. </li><li> Ajouter 20 pi de tampon de reconstitution dans chaque tube pour remettre en suspension les réactifs lyophilisés. </li><li> Mélanger par pipetage tampon de haut en bas 5 fois. Jetez un coup d&#39;oeil pour vous assurer que les réactifs lyophilisés sont complètement remis en suspension. La poudre blanche devrait disparaître dans le tube. </li><li> Ajouter 5 ul de matrice d&#39;ADN (de l&#39;étape 1 ou 2) pour les réactifs reconstitués et bien mélanger. </li><li> Fermer le tube et incuber à 60 ° C pendant 60 min dans un bain d&#39;eau ou d&#39;un bloc de chauffage. </li><li> Ajouter 5 ul de 10 x tampon de charge de gel pour terminer la réaction. </li><li> Charge 5 pi de réactionproduits à un gel d&#39;agarose à 2% coloré avec intercalation colorant d&#39;acide nucléique. </li><li> Exécutez gel à 100 V pendant 35 min dans un tampon 1x TBE. </li><li> Visualisez les résultats par la lumière UV. </li></ol> 6. Effectuer LAMP Réaction avec tampon de reconstitution contenant HNB ou fluorescent intercalant Dye <ol><li> Mélanger 125 ul de 10 x Pol tampon, 200 ul de 5 M triméthylglycine, 7,5 ul de 1 M MgSO 4, 7,5 pi d&#39;mM HNB 20 (ou 12,5 pi de 100x colorant fluorescent intercalant) et 660 ul (ou 655 pi) d&#39;eau à faire 1 ml de tampon de reconstitution. Mélanger par impulsions tourbillonnement pendant 10 secondes. </li><li> Utilisez le tampon reconstitué préparé à l&#39;article 6 de répéter les étapes 5.2 - 5.6. </li><li> Visualisez les résultats par l&#39;oeil nu (réaction contenant HNB) ou une lumière UV (réaction contenant colorant fluorescent intercalant). </li></ol> 7. Effectuer en temps réel LAMP Réaction avec Tube Scanner <ol><li> Ajouter 5 ul ADN à la reconstitutréactifs és contenant colorant fluorescent intercalant, fermer le tube et l&#39;insérer dans le scanner de tube. </li><li> Régler la température d&#39;incubation à 60 ° C. Mesurer la fluorescence à 520 nm pendant 60 min. </li></ol>

<ol>
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Les auteurs n&#39;ont rien à dévoiler.

Auparavant, nous avons développé un test LAMP sensible et spécifique ciblant l&#39;élément d&#39;insertion IS1111a 14. L&#39;élément IS1111 a été choisi parce qu&#39;il est hautement conservée entre les différentes souches de C. burnetii et son nombre élevé de copies (7-110) dans les bactéries (Klee 2006). Nos résultats ont montré que la lampe peut détecter environ 25 copies de l&#39;élément IS1111, qui peut en corrélation avec aussi peu que une copie ch...

Le petit Gram négatif bactérie Coxiella burnetii est l&#39;agent causal de la fièvre Q, qui est une zoonose dans le monde entier. En raison de la distribution mondiale de la fièvre Q et la forte infectivité de C. burnetii, les militaires et les civils américains déployés à l&#39; étranger sont à risque d&#39;être infectés dans les zones endémiques. La fièvre Q se manifeste sous deux formes: les infections aiguës et chroniques. La fièvre Q aiguë se présente avec des symptômes semblables à la grippe, l&#39;hépatite, ou la pneumonie, et est habituellement une maladie auto-limitation avec un faible taux de mortalité. La fièvre Q chronique, alors que moins répandue, se traduit souvent par une endocardite, qui a un taux de mortalité beaucoup plus élevé si non traitée 1,2. Par conséquent, le diagnostic précoce pour guider un traitement approprié est essentiel pour les soins aux patients. Réaction en chaîne par polymérase (PCR) et en temps réel de PCR quantitative (qPCR) essais ont été développés pour détecter C. ADN burnetii dans des cultures cellulaires et des échantillons cliniques 3-5 &lt;/ Sup&gt;. Les deux PCR et qPCR sont coûteuses et souvent pas facilement disponibles dans les zones de ressources limitées pour le travail de routine. Initialement décrit par Notomi 6, isothermes amplification de boucle médiée (LAMP) offre une méthode d&#39;amplification d&#39; ADN alternative. Lampe utilise l&#39; ADN polymerase Bst pour la synthèse d&#39;ADN par déplacement de brin ainsi que des ensembles d&#39; amorces spécialement conçues qui reconnaissent au moins six régions indépendantes du gène cible. L&#39;avantage le plus important de lampes est que l&#39;amplification se produit dans des conditions isothermes. Par conséquent, seul un bain d&#39;eau, bloc chauffant ou un incubateur est nécessaire. Cette méthode a été utilisée pour détecter plusieurs pathogènes rickettsies 7-9. La visualisation des produits d&#39;ADN amplifiés par électrophorèse sur gel est la méthode la plus précise qui permet de différencier les vrais positifs de faux positifs dus à une amplification non spécifique. Toutefois, les procédures impliquées dans l&#39;électrophorèse sur gel ne sont pas pratiques pour les ar des ressources limitéeseas. Plusieurs méthodes ont été développées pour détecter les produits de la réaction. Ces alternatives, telles que la turbidité dérivée de la formation de pyrophosphate de magnésium 10 ou à l&#39; aide d&#39; un colorant fluorescent intercalant pour être visualisé sous lumière UV 11,12 sont plus favorables que l&#39; exécution d&#39; un gel. Les réactifs LAMP ont été stables pendant un mois lorsqu&#39;il est conservé à 25 ° C et 37 ° C, qui sont des températures ambiantes dans les pays tropicaux et sub-tropicales où la fièvre Q est endémique 13. Un essai de lampe a été développé dans notre laboratoire pour détecter la présence de C. burnetii dans des échantillons de plasma 14. Nous décrivons ici un protocole simple pour la préparation du mélange réactionnel de lampe à partir des réactifs lyophilisés. Les réactifs lyophilisés sont stables pendant un mois lorsqu&#39;il est conservé à température ambiante. Lorsqu&#39;il est combiné avec une méthode de visualisation facile, ceci est une méthode idéale à utiliser pour détecter C. burnetii dans un cadre de ressources limitées.

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">LAMP primers</td>
			<td style="width:188px;">Eurofins MWG operon</td>
			<td style="width:131px;">10 nmol, salt free</td>
			<td style="width:240px;">Sequence designed by customer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Bst DNA polymerase</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">M0275L</td>
			<td style="width:240px;">8 units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Thermo pol buffer</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">B9004S</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">dNTP mixture</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">N0447L</td>
			<td style="width:240px;">10 mM each</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Betaine</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">B0300</td>
			<td style="width:240px;">5 M, Trimethylglycine</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Magnesium Sulfate</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">M3409-1ML</td>
			<td style="width:240px;">1 M</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Bluejuice</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">10816-015</td>
			<td style="width:240px;">gel loading buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">SYBR green</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">S7585</td>
			<td style="width:240px;">10,000X, visualize products in tubes</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">GelRed</td>
			<td style="width:188px;">Phenix Research Products</td>
			<td style="width:131px;">RGB-4103</td>
			<td style="width:240px;">10,000X, visualize products in gels</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Lyophilized reagents</td>
			<td style="width:188px;">Gene Reach</td>
			<td style="width:131px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Hydroxynaphthol blue</td>
			<td style="width:188px;">Fluka</td>
			<td style="width:131px;">33936-10G</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">ESEQuant tube scanner</td>
			<td style="width:188px;">Qiagen</td>
			<td style="width:131px;"></td>
			<td style="width:240px;">real-time detection</td>
		</tr>
	</tbody>
</table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

Coxiella burnetii, l&#39;agent responsable de la fièvre Q, est une bactérie intracellulaire obligatoire. Des dosages diagnostiques basées sur la PCR ont été développées pour détecter C. ADN burnetii dans des cultures de cellules et d&#39; échantillons cliniques. PCR nécessite un équipement spécialisé et une formation approfondie de l&#39;utilisateur final, et par conséquent, il ne convient pas pour le travail de routine en particulier dans une zone de ressources limitées. Nous avons mis au point une boucle à médiation par une amplification isotherme (LAMP) essai pour détecter la présence de C. burnetii dans des échantillons de patients. Ce procédé est effectué à une seule température d&#39;environ 60 ° C dans un bain-marie ou un bloc chauffant. La sensibilité de ce dosage de la lampe est très similaire à la PCR avec une limite de détection d&#39;environ 25 copies par réaction. Ce rapport décrit la préparation de la réaction en utilisant des réactifs lyophilisés et la visualisation des résultats en utilisant le bleu hydroxynaphthol (HNB) ou une lampe UV avec colorant fluorescent intercalant dans la réaction. Les réactifs LAMP étaient lyophilized et conservé à température ambiante (TA) pendant un mois, sans perte de sensibilité de détection. Ce test LAMP est particulièrement robuste car la préparation du mélange réactionnel ne comporte pas d&#39;étapes complexes. Cette méthode est idéale pour une utilisation dans des environnements à ressources limitées où la fièvre Q est endémique.

Les réactifs LAMP lyophilisés l&#39; intérieur du tube de 0,2 ml contient Bst ADN polymérase, des amorces et des dNTP. 20 ul de tampon de reconstitution est utilisée pour remettre en suspension les réactifs lyophilisés. La figure 1 montre les résultats de la réaction LAMP sur des gels d&#39; agarose avec des réactifs fraîchement préparés et des réactifs lyophilisés. Le réactif lyophilisé ne réduit pas son activité. Les réactions de LAMP prép...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Le développement de lyophilisé boucle médiées isothermes Amplification Réactifs pour la détection des<em&gt; Coxiella burnetii</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

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Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

10.4K vues.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Vidéo: The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

La détection de virus infectieux à partir terrain recueillies par les moustiques Assay culture cellulaire Vero

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Recherche

Immunologie et infection

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

Génération de Arenavirus recombinant pour le développement de vaccins approuvés par la FDA des cellules Vero

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

Évaluer le développement de cellules murines dendritiques plasmacytoïdes dans les plaques de Peyer Utiliser le transfert adoptif de progéniteurs hématopoïétiques

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

Analyse in vitro de la réponse immunitaire cellulaire Myd88 médiation au virus du Nil occidental souche mutante Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

Développement d&#39;un<em&gt; In vitro</em&gt; Système modèle pour l&#39;étude de l&#39;interaction de<em&gt; Equus</em&gt;<em&gt; Caballus</em&gt; IgE avec son récepteur de haute affinité FcsRI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

amplification isotherme (LAMP) Essais de boucle médiation pour la détection des espèces spécifiques de<em&gt; Eimeria</em&gt; Qui infectent les poulets

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Génération et multi-phénotypique criblage à haut contenu de<em&gt; Coxiella burnetii</em&gt; Mutants de transposon

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

Les analyses qualitatives et quantitatives pour la détection et la caractérisation des antimicrobiens en protéines

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Application Fluorescence Resonance Energy Transfer (FRET) pour examiner effecteur translocation efficacité par<em&gt; Coxiella burnetii</em&gt; Pendant siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

Développement d&#39;un plus sensible et spécifique chromogénique Agar moyen pour la détection de<em&gt; Vibrio parahaemolyticus</em&gt; Et autres<em&gt; Vibrio</em&gt; espèces

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Le développement de lyophilisé boucle médiées isothermes Amplification Réactifs pour la détection des

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